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A kind of Monascus purple ester synthase lip05, coding gene and application thereof

A technology of LIP05, encoding gene, applied in the field of genetic engineering, can solve problems such as no reports yet

Active Publication Date: 2020-10-16
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, Monascus has the ability to efficiently catalyze the synthesis of key flavor esters such as ethyl caproate, but so far, there is no report of an ester synthase from Monascus that can efficiently catalyze the synthesis of ethyl caproate and ethyl caprylate

Method used

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  • A kind of Monascus purple ester synthase lip05, coding gene and application thereof
  • A kind of Monascus purple ester synthase lip05, coding gene and application thereof
  • A kind of Monascus purple ester synthase lip05, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Cloning of the gene encoding ester synthase LIP05

[0044] 1 Culture of Monascus purple

[0045] Under aseptic conditions, Monascus purple was inoculated into a 300mL Erlenmeyer flask containing 100mL of fermentation medium, and cultured on a shaker at 30±1°C and 150±10r / min for 4-8d. The fermentation medium is composed of glucose 70g / L, soybean cake powder 10g / L, MgSO 4 0.20g / L, NaNO 3 2.0g / L, NaH 2 PO 4 1.0g / L, adjust the pH to 4.5, and sterilize at 115°C for 20min.

[0046] 2 Extraction of Monascus purple total RNA

[0047] The above-mentioned Monascus violaceum cultured for 6 days was sampled, and the total RNA was extracted using the BIOMIGA Fungal RNA Extraction Kit. Specific steps are as follows:

[0048] (1) Weigh 100mg of fungal culture into a 1.5mL or 2.0mL centrifuge tube, freeze in liquid nitrogen, grind the fungus into powder with a grinding pestle (if possible, put the tissue in a mortar, and grind it into powder with liquid nitrogen , quickly t...

Embodiment 2

[0085] Construction of Escherichia coli engineering strain expressing ester synthase LIP05

[0086] Linearization of 1pCold-TF vector

[0087] Primers were designed to linearize the pCold-TF vector by PCR. The primers were designed as follows: Forward primer: 5'-ctgcagtctagataggtaatc-3' (SEQ ID No.5)

[0088] Reverse primer: 5'-accaccactacctcgtggca-3' (SEQ ID No.6)

[0089] The PCR reaction system is shown in Table 4.

[0090] Table 4 PCR reaction system of linearized vector pCold-TF

[0091] Reagent Volume (μl) wxya 2 o

21.0 dNTP Mixture (2.5mM each) 3.0 10×Ex Taq Buffer 3.0 Forward primer (10μM) 0.6 Reverse primer (10μM) 0.6 pCold-TF vector 0.8 Q5 DNA Polymerase (5U / μl) 1.0 total 30.0

[0092] The PCR amplification reaction procedure is as follows:

[0093]

[0094] The amplified DNA band is used for plasmid construction after DNA purification.

[0095] 2 Plasmid construction

[0096] pass II car...

Embodiment 3

[0103]Synthesis of Ethyl Hexanoate and Ethyl Octanoate Catalyzed by Ester Synthase LIP05 in Liquid Aqueous Phase System

[0104] Induced expression of 1 ester synthase LIP05

[0105] Pick the verified correct transformants, transfer them to LB liquid test tubes containing appropriate ampicillin antibiotics, culture overnight at 37±1°C, then inoculate a 300mL Erlenmeyer flask containing 100mL LB medium with an inoculum of 1% (v / v), shake The bed was cultured at 37±1° C. and 200 rpm for 3 hours, then the inducer IPTG with a final concentration of 0.5 mM was added, and cultured at 20±1° C. and 200±10 rpm for 20 hours.

[0106] 2 Preparation of crude enzyme solution of ester synthase LIP05

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Abstract

The invention provides ester synthetase LIP05 of monascus purpureus, an encoding gene and application thereof and belongs to the technical field of gene engineering. The amino acid sequence of the ester synthetase LIP05 of monascus purpureus is as shown in SEQ ID No.1. The nucleotide sequence of the encoding gene of the ester synthetase LIP05 of monascus purpureus is as shown in SEQ ID No.2. The ester synthetase LIP05 provided by the invention is lipase, reported for the first time, which has the characteristic of catalytically synthesizing ethyl hexanoate and ethyl caprylate simultaneously in a water phase system. Through recombinant expression of escherichia coli, an obtained crude enzyme solution has the ability of catalytically synthesizing ethyl hexanoate and ethyl caprylate simultaneously in the water phase system effectively, so that the ester synthetase LIP05 provided by the invention can be used for catalytically synthesizing important flavor ester ethyl hexanoate and ethyl caprylate in the water phase system in Baijiu brewing.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a monascus purple ester synthase LIP05, an encoding gene and an application thereof. Background technique [0002] The main substances of Luzhou-flavor liquor are water and ethanol, and a small amount of flavor substances such as esters, acids, ketones, etc., although their content only accounts for 1% to 3% of the total body of the liquor, it is the soul of the liquor. Product organoleptic and flavor. Among them, ethyl caproate and ethyl octanoate are important esters that determine the quality of Luzhou-flavor liquor. In actual production, it has become a consensus in the industry to regard the content of important esters such as ethyl caproate in the wine body as an important parameter to measure product quality. However, due to the slow production of ester aroma during the brewing process, that is, the low synthesis efficiency of ethyl caproate, ethy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12N15/70C12N1/21C12N15/55C12P7/64C12N15/11C12R1/645C12R1/19
CPCC12N9/20C12N15/11C12N15/70C12P7/6436C12Y301/01003
Inventor 李秀婷徐友强孙宝国王晓程
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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